The objective of this proposal is to investigate specific biochemical modifications of liver pyruvate kinase (L-type isozyme) elicited in response to dietary or hormonal stress. One specific modification being investigated is the phosphorylation of liver pyruvate kinase in response to cAMP-dependent, cAMP-independent and calcium activated mechanisms. The second modification being investigated is the role of protein synthesis and catabolism of liver pyruvate kinase as a mechanism to control the catalytic activity in vivo in response to dietary (high carbohydrate diet or fasting) or hormonal stress in streptozotocin or alloxan diabetic rats. The studies on the role of phosphorylatton will be carried out by incubation of hepatocytes with 32P in the presence or absence of hormones (glucagon, catecholamines and insulin) or effectors (cAMP, cGMP and calcium). The 32P-liver pyruvate kinase will be isolated by quantitative immunoprecipitation with goat-anti-liver pyruvate kinase. In each case the stoichiometry of 32P incorporation into the enzyme will be determined. Furthermore, in each instance the location of the 32P in the primary sequence of the enzyme will be determined by tryptic peptide mapping and autoradiography. In the studies on the role of pyruvate kinase synthesis and catabolism, the relative rates of synthesis and apparent rates of degradation of liver pyruvate kinase will be determined in vivo through the isolation of isotopically labeled liver pyruvate kinase by quantitative immunoprecipitation at specific times after a single i.p. injection of (3H)-leucine into rats in various nutritional or hormonal states.